Báo cáo khoa học: The molecular identity of the mitochondrial Ca2+ sequestration system potx

MINIREVIEW
The molecular identity of the mitochondrial Ca
2+
sequestration system
Anatoly A. Starkov
Weill Medical College of Cornell University, New York, NY, USA
The standard model
The ability to accumulate, retain and release Ca
2+
is a
fundamental ubiquitous function of animal mitochon-
dria. Extensive research during the last 50 years has
resulted in a consensus model of mitochondrial Ca
2+
handling that adequately accommodates most if not all
experimental data, referred to here as ‘the standard
model of mitochondrial Ca
2+
handling’. This model is
shown in Fig. 1 in a greatly simplified form, and
assumes that mitochondria accumulate exogenous
Ca
2+
by means of an electrogenic carrier that facili-
tates Ca
2+
transport across the inner mitochondrial
membrane (IM) into the matrix. The transport is cou-
pled to simultaneous accumulation of inorganic phos-
phate. Inside the matrix, accumulated Ca
2+

and
phosphate are stored in the form of osmotically inac-
tive precipitates, and eventually are slowly released
back into the cytosol with the assistance of
Ca
2+
⁄ nNa
+
and ⁄ or Ca
2+
⁄ 2H
+
exchangers (Fig. 1)
that are also situated in the IM. When accumulated
above a certain threshold, Ca
2+
triggers opening of
the so-called permeability transition pore (PTP). This
may also be mediated by matrix proteins such as
cyclophilin D (CypD). Opening of the PTP is thought
to have a detrimental effect on mitochondria and cell
well-being in general. The Ca
2+
uniporter system and
the PTP structure are thought to consist of proteins,
but the molecular identities of these proteins are
unknown. The only two Ca
2+
transport-related pro-
teins that have been identified are the Ca

2+
⁄ nNa
+
exchanger and Ca
2+
⁄ 2H
+
exchanger: the gene for the
CGP37157-sensitive mitochondrial Ca
2+
⁄ nNa
+
exchanger has recently been identified as NCLX
Keywords
brain mitochondria; Ca
2+
accumulation; Ca
2+
and Pi precipitate; calciphorin; calcium
uniporter; calvectin; dense granules; gC1qR;
liver mitochondria; permeability transition
pore
Correspondence
A. A. Starkov, 1300 York Ave A501, New
York, NY 10065, USA
Fax: +1 212 746 8276
Tel: +1 212 746 4534
E-mail:
(Received 8 March 2010, revised 23 May
2010, accepted 23 June 2010)

doi:10.1111/j.1742-4658.2010.07756.x
There is ample evidence to suggest that a dramatic decrease in mitochon-
drial Ca
2+
retention may contribute to the cell death associated with
stroke, excitotoxicity, ischemia and reperfusion, and neurodegenerative dis-
eases. Mitochondria from all studied tissues can accumulate and store
Ca
2+
, but the maximum Ca
2+
storage capacity varies widely and exhibits
striking tissue specificity. There is currently no explanation for this fact.
Precipitation of Ca
2+
and phosphate in the mitochondrial matrix has been
suggested to be the major form of storage of accumulated Ca
2+
in mito-
chondria. How this precipitate is formed is not known. The molecular iden-
tity of almost all proteins involved in Ca
2+
transport, storage and
formation of the permeability transition pore is also unknown. This review
summarizes studies aimed at identifying these proteins, and describes the
properties of a known mitochondrial protein that may be involved in Ca
2+
transport and the structure of the permeability transition pore.
Abbreviations
ANT, adenine nucleotide translocase; CGP37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; CypD, cyclophilin D;

EKR, extracellular-signal-regulated kinase; Hrk, a product of harakiri gene; IM, inner mitochondrial membrane; PTP, permeability transition
pore; Ru360, C
2
H
26
Cl
3
N
8
O
5
Ru
2
; smARF, ‘‘short mitochondrial ARF’’, a short isoform of p19ARF protein.
3652 FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS
(SLC24A6 family) [1], and that for the Ca
2+
⁄ 2H
+
exchanger has been identified as Letm1 [2]. These two
proteins will not be reviewed here; please see the
review by Chinopoulos & Adam-Vizi [3] and that by
Pivovarova & Andrews [4] in this issue for details.
Nevertheless, extensive studies to identify proteins
involved in Ca
2+
transport, storage and the PTP have
been performed over the last 50 years. This review is
not concerned with kinetic, biophysical channel-
related, bioenergetic or pathophysiological aspects of

Ca
2+
handling in mitochondria; numerous excellent
reviews on these subjects can be found elsewhere.
Here, the present review describes some of the most
prominent and followed-up research efforts to identify
the proteins involved in Ca
2+
transport, storage and
PTP, and presents a hypothesis on this subject that
somewhat modifies the ‘standard model’.
Ca
2+
uniporter system
Although the molecular identity of the mitochondrial
calcium uniporter is still unknown, experimental data
have suggested that it is a highly selective, inward-
rectifying ion channel [5], a ‘gated’ pore containing a
Ca
2+
binding site on the cytosolic side of the inner
mitochondrial membrane that activates Ca
2+
trans-
port [6,7]. It has been suggested that mitochondrial
calcium uniporter contains at least two subunits, one
of which is a dissociable intermembrane factor that is
glycoprotein in nature, and that the mitochondrial
calcium uniporter is regulated by association and dis-
sociation of this factor, activated by calcium binding

[8]. It has been shown that mitochondria depleted of
endogenous Ca
2+
exhibited low initial rate of energy-
dependent Ca
2+
uptake. Pre-incubation of de-ener-
gized mitochondria with added Ca
2+
stimulated their
energy-dependent Ca
2+
uptake up to 10-fold, with
strong cooperativity in the velocity–substrate curves for
Ca
2+
-depleted mitochondria. To explain these and
other kinetic peculiarities of Ca
2+
transport, a model
has been proposed in which the Ca
2+
-transporting
system is present in a de-activated state in the absence
of cytosolic Ca
2+
, and formation of the active Ca
2+
uniporter is triggered by an increase in external Ca
2+

.
The uniporter is formed by oligomerization of two
or more protomers, resulting in formation of the
ruthenium- and lanthanides-sensitive Ca
2+
-conducting
gated channel [9].
As already mentioned, the molecular identity of the
mitochondrial calcium uniporter remains unknown,
despite considerable efforts by many prominent
researchers. Since the pioneering reports of Sottocasa
et al. [10] and Lehninger [11], numerous attempts have
been made to isolate the calcium uniporter [12–23].
Various Ca
2+
binding proteins and peptides have been
isolated and characterized, all of which are able to
bind Ca
2+
in a ruthenium red- and La
3+
-inhibited
fashion, and some of which are able to transport
bound Ca
2+
through artificial bilayer membranes.
Reviewing all this literature is beyond the scope of the
present review: Lars Ernster’s [23a] and Saris and
Carafoli’s [24] recent review provide comprehensive lit-
erature surveys on the history of Ca

2+
transport and
attempts to isolate the Ca
2+
uniporter. The present
review covers only the most followed-up and detailed
studies.
Fig. 1. Standard model of mitochondrial Ca
2+
handling. Mitochon-
dria accumulate exogenous Ca
2+
by means of an electrogenic car-
rier (calcium uniporter, ‘U’) that facilitates Ca
2+
transport across the
inner mitochondrial membrane (IM) into the matrix. The transport is
coupled to simultaneous accumulation of inorganic phosphate (not
shown). Inside the matrix, accumulated Ca
2+
and phosphate are
stored in the form of osmotically inactive precipitates (‘precipitate’),
and eventually slowly released back into the cytosol through a
Ca
2+
⁄ nNa
+
(not shown) and ⁄ or a Ca
2+
⁄ 2H

+
exchanger that is also
located in the IM. The process of Ca
2+
uptake is driven by the
membrane potential; the process of Ca
2+
release is driven by the
pH gradient, in the case of the Ca
2+
⁄ 2H
+
exchanger. Elevated
intramitochondrial Ca
2+
can stimulate the activities of enzymes of
the tricarboxylic acid cycle (TCA), thereby boosting energy produc-
tion in the mitochondria. When it accumulates above a certain
threshold, Ca
2+
triggers PTP opening, and this is also modulated by
matrix-located protein cyclophilin D (CypD). E, exchanger; RC,
respiratory chain.
A. A. Starkov Mitochondrial Ca
2+
sequestration system
FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS 3653
Calvectin
The earliest extensively studied preparations of mito-
chondrial Ca

2+
binding protein(s) were isolated by
Sottocasa et al. from intermembrane space of rat liver
mitochondria [10] and ox liver mitochondria [14].
These preparations were capable of high-affinity Ca
2+
binding that was inhibited by ruthenium red and
La
3+
. These preparations showed a single band of
approximately 30 kDa on PAGE, and contained sialic
acid and neutral and amino sugars, typical of glyco-
proteins, a high content of dicarboxylic amino acids,
and some bound Ca
2+
and Mg
2+
. This preparation
was capable of binding Ca
2+
with high affinity (K
d
of
approximately 100 nm), and also contained a number
of low-affinity Ca
2+
binding sites. It was named ‘cal-
vectin’ [25], and was suggested to represent the mito-
chondrial Ca
2+

carrier or a major component thereof.
Similarly isolated glycoprotein increased the conduc-
tance of artificial lipid bilayers in the presence of
Ca
2+
, and the conductance was sensitive to ruthenium
red [22], implying that it may be the Ca
2+
uniporter
or part thereof. Further studies revealed a set of
unique features for this preparation. One of them was
that the glycoprotein was found primarily in the inter-
membrane space in both a free soluble form and also
tightly bound to the inner membrane, but was absent
in the matrix of mitochondria [26]. Binding to inner
and outer membranes apparently required Mg
2+
and ⁄ or Ca
2+
[27]. Moreover, calvectin appeared to be
able to move reversibly between mitochondrial com-
partments in the presence of Ca
2+
. The binding to
the membrane could further be modulated by pyridine
nucleotides, which also bind to calvectin; bound
NAD+ decreased the association of calvectin with the
membrane [28]. Mitochondria could be depleted of cal-
vectin by treating them with uncoupling concentrations
of pentachlorophenol in the presence of phosphate and

acetate. This treatment affected the ability of mito-
chondria to release pre-loaded Ca
2+
in response to the
addition of pentachlorophenol, with an almost linear
correlation between the amount of released glycopro-
tein and the rate of Ca
2+
efflux [29]. Adding the glyco-
protein back to mitoplasts (mitochondria stripped of
their outer membrane) depleted of it by swelling in
oxaloacetate ⁄ EDTA restored the Ca
2+
uptake if
Mg
2+
was also included in the mixture [28]. Antibod-
ies raised against calvectin were able to inhibit Ca
2+
transport in mitoplasts, indicating that this glycoprotein
is a required part of the mitochondrial Ca
2+
transport
machinery [30], (to note, a review by Saris and Carafoli
mentions that ‘‘Saris found that the antiserum formed
four precipitation bands in Ouchterlony immunodiffu-
sion tests and did not inhibit Sr
2+
uptake by the unipor-
ter’’ [24]. We were not able to find another published

record of that finding which is important because mito-
chondria are known to accumulate both Ca
2+
and Sr
2+
with about similar efficiency and ruthenium red sensitiv-
ity. Hence, this finding might imply that a conformation
of the ‘‘uniporter’’ that transports Ca
2+
is different
from that transporting Sr
2+
). The authors suggested an
interesting but rather simple ‘two-step’ model of
calvectin involvement in Ca
2+
transport: first, soluble
calvectin in the intermembrane space binds Ca
2+
and
associates spontaneously with the inner membrane; sec-
ond, it carries Ca
2+
through the membrane and some-
how returns back to the outer surface of the inner
membrane [31]. Eventually, a single protein was purified
from these crude preparations that migrated at approxi-
mately 14 kDa on SDS ⁄ PAGE and had a minimum
molecular weight of 15 577 calculated on the basis of its
amino acid composition. However, no sugars were

found in this protein, although it had a high content of
glutamic and aspartic acids. This protein also carried
fewer low-affinity Ca
2+
binding sites than the original
‘calvectin’ [23a].
Calciphorin
An integral low-molecular-weight membrane protein
with the properties of a Ca
2+
ionophore was isolated
from calf heart inner mitochondrial membrane [15–
17,32,33]. It was characterized as a 3000 Da high-affin-
ity calcium carrier and named ‘calciphorin’ [16]. In
contrast to hydrophilic calvectin, the calciphorin was
hydrophobic and lacked phospholipids, sugars and free
fatty acids. Calciphorin was able to extract Ca
2+
into
an organic solvent phase and to transport Ca
2+
through a bulk organic phase in the presence of a lipo-
philic anion (picrate), indicating the electrophoretic
nature of the calciphorin–Ca
2+
complex. The Ca
2+
extraction was strongly inhibited by ruthenium red
and lanthanum. The selectivity of ion extraction
by calciphorin was Zn

2+
>Ca
2+
,Sr
2+
>Mn
2+
>
Na
+
>K
+
[32]. The Ca
2+
binding site had a dissoci-
ation constant of 5.2 pm, with 1 mole Ca
2+
bound per
mole of calciphorin [32]. Calciphorin was shown to
transport Ca
2+
in a lipid bilayer membrane model
such as reconstituted phospholipid vesicles. Further-
more, calciphorin-mediated Ca
2+
transport across the
vesicle membrane was toward the negatively charged
side of the membrane. This calciphorin-mediated cal-
cium transport in vesicles was also strongly inhibited
by ruthenium red and La

3+
[33].
The role of calciphorin as the Ca
2+
ionophore was
subsequently challenged by Sokolove and Brenza [34],
Mitochondrial Ca
2+
sequestration system A. A. Starkov
3654 FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS
who isolated a mixed protein–lipid fraction from rat
liver mitochondria that had properties similar to those
of calciphorin. They attributed all the Ca
2+
-binding
and transporting ability of that fraction to its lipid
components. In another study, these authors demon-
strated that cardiolipin binds Ca
2+
with high affinity
(apparent K
d
= 0.70 ± 0.17 lm) and can extract Ca
2+
into a bulk organic phase. The interaction of cardioli-
pin with Ca
2+
was insensitive to Na
+
, but was inhib-

ited by divalent cations (Mn
2+
>Zn
2+
>Mg
2+
). In
addition, La
3+
and ruthenium red were found to be
strong inhibitors of Ca
2+
binding by cardiolipin [35].
However, it should be noted that the isolation proce-
dure used by Sokolove and Brenza was similar but not
identical to that originally reported by Shamoo’s group
who later successfully isolated ‘calciphorin’ from rat
liver mitochondria [36]. Nevertheless, it is still not clear
whether ‘liver calciphorin’ and ‘heart calciphorin’ are
the same proteins, or indeed whether the procedure
described by Jeng and Shamoo is reproducible. As can
be seen from Table 1 in [36], the ‘calciphorin’ isolated
from liver and two ‘calciphorin’ isolates from calf heart
were quite different in terms of their estimated molecu-
lar mass and other parameters. To the best of our
knowledge, there were no new reports on calciphorin
after 1984.
Mironova’s glycoprotein and peptide
Mironova’s group worked on isolation and identifica-
tion of Ca

2+
-transporting substances in mitochondria
for almost two decades since approximately 1976, but
most of the earlier results were published in hard-to-
access Russian journals. The authors isolated a compo-
nent capable of inducing selective Ca
2+
transport in
artificial bilayer lipid membranes from mitochondria
and homogenates of various animal and human tissues.
The Ca
2+
-transporting properties of this component
were ascribed to the presence of a glycoprotein and a
peptide. The 40 kDa glycoprotein and 2 kDa peptide
from beef heart homogenate and mitochondria induced
highly selective Ca
2+
transport through bilayer lipid
membranes. The glycoprotein contained 60–70% and
30–40% protein and carbohydrate, respectively. Sulfur-
containing amino acids (1 mole per 1 mole of glycopro-
tein) and sialic acids (2 or 3 moles per 1 mole of glyco-
protein) were also detected in the glycoprotein, and it
was enriched in asparagine and glutamine [21], similar
to calvectin. Lipids were not essential for the Ca
2+
-
transporting activity of glycoprotein. Micromolar con-
centrations of the glycoprotein and the peptide were

found to greatly increase the conductivity of bilayer
lipid membranes. Ruthenium red abolished the glyco-
protein- and peptide-induced Ca
2+
transport in bilayer
lipid membranes. A transmembrane Ca
2+
gradient
induced an electric potential difference whose magni-
tude was close to the theoretical value for optimum
Ca
2+
selectivity. The authors also identified thiol
groups that were essential for Ca
2+
transport in both
the glycoprotein and the peptide. On the basis of these
studies, the authors proposed a model in which the
peptide is an active Ca
2+
-transporting portion of the
glycoprotein, which lacks Ca
2+
-transporting activity
when the peptide is detached. Ca
2+
moves through spe-
cial channels in the membrane formed by the peptide,
and the glycoprotein, which has many Ca
2+

-binding
centers, creates a high concentration of Ca
2+
near the
channel mouth. Functioning of the channels is con-
trolled by thiol-disulfide transitions of sulfur-containing
groups of the glycoprotein–peptide complex [21].
A decade later, the same group (in collaboration with
Saris) generated polyclonal rabbit antibodies against a
‘Ca
2+
-binding mitochondrial glycoprotein’ (presumably
the former glycoprotein). These antibodies were found
to inhibit the uniporter-mediated transport of Ca
2+
in
mitoplasts prepared from rat liver mitochondria. Sper-
mine, a modulator of the uniporter, decreased the inhi-
bition [37]. The peptide was isolated and purified to
homogeneity and shown to form a Ca
2+
-transporting
channel in bilayer lipid membranes, requiring addition
of the peptide from both sides of the membrane, [20].
This suggested that the channel is formed by two or
more subunits, as in formation of the gramicidin D
channel [38]. The authors also demonstrated that the
Ca
2+
-binding 40 kDa glycoprotein previously reported

as a precursor of the peptide may in fact be an irrele-
vant contaminant, as it was immunologically indistin-
guishable from beef plasma orosomucoid protein.
However, antibody raised against the orosomucoid was
not able to inhibit mitochondrial Ca
2+
uptake [20], in
contrast to the antibodies derived against mitochon-
drial glycoprotein in the previous study [37]. Never-
theless, the authors concluded that the presence of the
40 kDa glycoprotein in association with a channel-
forming peptide [39] was due to co-purification.
Most recent ‘Ca
2+
uniporter’ isolations
Chavez’s group isolated a semi-purified extract of pro-
teins from rat kidney cortex mitochondria that con-
ferred Ca
2+
-transporting capacity to energized
cytochrome oxidase-containing proteoliposomes, and
generated a mouse hyperimmune serum that inhibited
Ca
2+
transport in mitoplasts and proteoliposomes.
The serum recognized three major proteins of 75, 70
and 20 kDa. The purified antibody recognizing the
A. A. Starkov Mitochondrial Ca
2+
sequestration system

FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS 3655
20 kDa component inhibited Ca
2+
transport by
approximately 70% in mitoplasts, suggesting that this
20 kDa protein is a necessary component of the Ca
2+
uniporter [23]. In a follow-up study, the same group
isolated an 18 kDa protein that binds Ru360 (an
inhibitor of Ca
2+
uniporter) with high affinity, and
proposed that it is part of the uniporter [40]. Most
recently, these authors isolated a Ca
2+
-transporting
protein fraction and separated it further by preparative
electrofocusing. After incorporating the separated frac-
tions into cytochrome oxidase containing proteolipo-
somes, they recovered two Ca
2+
-transporting
activities, only one of which was inhibited by Ru360.
On the basis of these results, the authors suggested
that the Ca
2+
uniporter is composed of at least two
different subunits that become partially dissociated at
low pH. The Ru360-resistant proteins are dissociated
at low pH and represent the Ca

2+
channel, whereas
the subunit that binds to Ru360 remains linked to the
channel at higher pH [41]. The same group also
showed that glycosyl residues on the putative Ca
2+
uniporter are not required for Ca
2+
transport activity:
deglycosylation of mitoplasts using glycosidase F
removed the ruthenium red sensitivity of Ca
2+
uptake
but did not inhibit it [42].
It is very surprising that so much effort spanning
several decades of research did not result in molecu-
lar identification of any of the isolated putative
Ca
2+
-transporting proteins. Even the most recent stud-
ies by Zazueta et al. [40], performed when the majority
of the new proteomics approaches, sequencing tech-
niques and a wealth of genetic information were
already available, did not identify the isolated proteins.
Unfortunately, the chances of reproducing the older
research and isolating the same proteins are low. Pro-
tein purification from mitochondrial membranes that
carry hundreds of proteins is akin to magic: unless a
spell is cast precisely (in this case a step-by-step isola-
tion protocol listing all the reagents, procedures and

conditions), the result could be just a sore throat. It
may be more productive to adopt a targeted approach,
selecting a few known mitochondrial proteins fitting
the required ‘profile’ and using genetic approaches to
prove their involvement in Ca
2+
transport. What kind
of ‘profile’ for a putative Ca
2+
uniporter can be
deduced from these older studies? The structure of this
protein should accommodate the following features:
the protein should be of moderate to low molecular
mass, approximately 15–40 kDa, it should be capable
of binding Ca
2+
, the binding should be inhibited by
ruthenium red and other known Ca
2+
uniporter
inhibitors, it also should be able to bind to the inner
mitochondrial membrane from at least the cytosolic
side, preferably in the presence of Ca
2+
(like calvec-
tin), and, according to all the hypotheses reviewed
above and a wealth of other known characteristics
regarding Ca
2+
transport, should be able to form a

gated pore comprising several identical protomers or
as a complex with other proteins. A known protein
that mostly fits this profile is discussed below.
Storage of Ca
2+
in mitochondria
Net Ca
2+
uptake into mitochondria requires co-trans-
port of an IM-permeable anion such as acetate or
phosphate. In the latter case, the accumulated Ca
2+
forms a precipitate in the matrix of mitochondria in an
apparently spontaneous process. The precipitate can
store large amounts of Ca
2+
and is readily observed in
isolated mitochondria by electron microscopy [4,43].
The precipitates appear in the form of large (50–
100 nm diameter) electron-dense granules with a hol-
low electron transparent core, and are always found in
immediate proximity to the IM [43]. Formation of the
Ca
2+
and phosphate precipitates is thought to be the
major mechanism of Ca
2+
storage in mitochondria
[4,43,44]. It has been suggested that a protein or other
matrix constituents may serve as a nucleation center

facilitating formation of the Ca
2+
precipitates [43].
Indeed, the presence of a protein may explain the
always amorphous nature of Ca
2+
-phosphate precipi-
tates, which is somewhat puzzling because hydroxyapa-
tite [Ca
5
(PO4)
3
(OH)], the most commonly found
composition of mitochondrial Ca
2+
and Pi precipi-
tates, is crystalline. In blood, where high levels of
Ca
2+
and Pi are standard, a protein called ‘fetuin’ had
been shown to inhibit the precipitation of hydroxyapa-
tite from supersaturated solutions of calcium and
phosphate [45]. Perhaps a similar protein serves the
same function in the mitochondrial matrix. The pres-
ence of substantial amounts of the Ca
2+
-binding pro-
teins mitocalcin [46], calbindin-28k and calbindin-30k
(calretinin) in a particulate fraction of rat brain [47]
and in brain mitochondria [48,49] has been demon-

strated previously, and annexin I [50] and annexin VI
[51] were found in liver mitochondria. At least some of
these proteins (annexin VI) serve as nucleation factors
in vitro [52]. However, the contribution of these pro-
teins to mitochondrial Ca
2+
storage has not been
examined. Although Ca
2+
-binding matrix-located pro-
teins are the main candidates for the role of nucleation
factors, non-protein factors such as mitochondrial
DNA cannot be ruled out, as the Ca
2+
-binding ability
of DNA is well known [53].
The Ca
2+
and phosphate granules have been iso-
lated from Ca
2+
-loaded rat liver mitochondria and
Mitochondrial Ca
2+
sequestration system A. A. Starkov
3656 FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS
their composition assessed [54]. The granules contained
significant amount of carbon and nitrogen, indicating
the presence of protein(s). However, the protein was
not isolated or identified because the focus of that

study was identifying the molecular form of the Ca
2+
and phosphate precipitate. In addition, protein identifi-
cation techniques were much more time-consuming
and costly in 1967 when the study was performed than
they are now. The Ca
2+
-phosphate precipitates are
discussed in more detail elsewhere [3].
Proteins implicated in PTP formation
One of the most dramatic manifestations of abnormal
Ca
2+
homeostasis in mitochondria is the opening of a
large channel called the ‘permeability transition pore’
(PTP) in the inner membrane that renders them inca-
pable of energy production and can result in cell death
by either apoptosis or necrosis. The functional and
physiological aspects of PTP and mitochondrial Ca
2+
transport have been reviewed extensively [55–61].
After a certain tissue-dependent threshold for the
accumulated Ca
2+
is reached, the permeability of IM
to solutes abruptly increases due to opening of the
PTP, a protein-mediated pore in the IM. It has been
suggested that opening of the PTP is probably trig-
gered by the increase in the free matrix Ca
2+

concen-
tration [55–61], although the evidence for this is
ambiguous. The free matrix Ca
2+
does increase some-
what upon loading mitochondria with Ca
2+
, but does
not exhibit any abrupt changes immediately before
PTP opening [44]. On the other hand, it increases sig-
nificantly upon loading of brain mitochondria with
Ca
2+
if opening of the PTP is inhibited [44]. There-
fore, it is not clear whether opening of the PTP is trig-
gered by the free matrix Ca
2+
or bound matrix Ca
2+
,
or both together. In either case, factors affecting for-
mation of the Ca
2+
and phosphate precipitates are
expected to influence the Ca
2+
threshold for PTP acti-
vation. Changes in the precipitation characteristics are
expected to have a strong effect on the overall Ca
2+

retention and storage in mitochondria.
The molecular identity of the protein(s) that actually
form the PTP channel remains a mystery. Past studies
identified several proteins involved in the PTP forma-
tion or modulation, such as the voltage-dependent
anion channel, the adenine nucleotide translocator
(ANT), and, more recently, the mitochondrial phos-
phate transporter PIC [59], although none of these
proteins are currently thought to directly form the
PTP channel [59,60]. Until recently, mitochondrial
ANT was viewed as the most likely PTP-forming pro-
tein [57,59,60]. ANT may also interact with another
matrix protein, cyclophilin D (CypD) [62]. The latter is
a target of cyclosporin A, a peptide inhibitor of PTP.
Although the role of CypD in modulating the Ca
2+
threshold for PTP activation had recently been
strongly confirmed [63–66], the role of ANT in PTP
formation was strongly challenged [67]. The PTP in
mitochondria isolated from CypD-ablated mice is
insensitive to cyclosporin A and exhibits a much
higher Ca
2+
threshold [63–66], whereas the PTP is
activated by Ca
2+
accumulation in ANT-deficient liver
mitochondria isolated from mice that were genetically
ablated of ANT in the liver [67].
Experimental evidence supporting a role for the volt-

age-dependent anion channel in PTP formation has
been discussed in detail [60,61]. However, the recent
finding that genetic ablation of any of the three mam-
malian voltage-dependent anion channel isoforms or
all of them together does not affect Ca
2+
-induced PTP
opening strongly suggests that voltage-dependent anion
channels are not involved in PTP channel formation
[68]. While the molecular identity of the PTP channel-
forming protein remains unknown, a role for the mito-
chondrial phosphate transporter PIC in PTP formation
cannot be ruled out [59].
An alternative model of PTP implicates no specific
proteins in the role of the PTP channel [69]. According
to this model, the pore is formed by aggregation of
some misfolded integral membrane proteins; transport
through these proteins is normally be blocked by
cyclophilin D or other chaperones but Ca
2+
accumula-
tion and or oxidative stress increase the number of
misfolded proteins. When the number of protein clus-
ters exceeds the number of chaperones available to
block transport, opening of ‘unregulated pores’ that
are no longer sensitive to PTP inhibitors such as cyclo-
sporin A would occur [69]. Although interesting, this
model fails to account for approximately half of the
known PTP features, such as its fast reversibility by
Ca

2+
chelation, its sensitivity to regulation by matrix
pH, transmembrane voltage, fixed pore size, etc. [60].
Overall, literature analysis [55–61] allow us to for-
mulate a minimum set of requirements to be fulfilled
by a plausible candidate for the role of PTP channel-
forming protein. First, it has to be able to bind to the
IM. Although it has always been presumed that the
PTP-forming protein is an integral protein embedded
in the IM, there is no evidence to support this pre-
sumption. The PTP-forming protein does not have to
be located in the IM before it forms a channel; it may
simply bind to the IM and move into the IM upon
activation. There are numerous examples of proteins
moving between various cellular compartments and
membranes upon activation. Second, it has to be able
A. A. Starkov Mitochondrial Ca
2+
sequestration system
FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS 3657
to form a large (approximately 2–3 nm diameter)
transmembrane channel to allow the passage of
charged and uncharged solutes up to 1500 Da. Third,
it has to be able to form the channel in a fully revers-
ible, fast and Ca
2+
-dependent fashion, as the full
reversibility of PTP opening upon Ca
2+
chelation and

its fast transition between an open and a closed state
are well known [55]. Finally, the molecular structure of
this putative protein should ideally feature Ca
2+
-bind-
ing sites, reactive thiol groups to facilitate channel for-
mation upon oxidation, and conformationally critical
b-sheets, as suggested by the effect of cyclophilin D,
which is a peptidyl-prolyl-cis ⁄ trans isomerase.
The above features are a minimum set of features
that, if present in a single protein, would strongly
implicate it as a plausible candidate for the role of
PTP channel-forming protein. Other PTP features such
as regulation by adenine nucleotides and effectors of
ANT may be due to other proteins that interact with
this putative PTP channel and modulate its activity.
gC1qR
Although there may be a number of unknown mito-
chondrial proteins that fulfil these requirements, at
least one ubiquitous and evolutionary conserved
eukaryotic protein, gC1qR, meets these requirements
in full. As mentioned earlier, the molecular identity of
the protein(s) that actually form the PTP channel
remains the most intriguing question. On the basis of
structural and other information, we hypothesize that
the gC1qR protein, also known as p32, gC1QR ⁄ 33,
splicing factor 2 (SF2) and hyoluronan-binding pro-
tein 1 (HABP1), is the most plausible candidate for the
role of PTP channel-forming protein. gC1qR is a 23.8.
kDa multifunctional cellular protein (although it

migrates at 33 kDa in SDS ⁄ PAGE, probably due to
glycosylation and strong charges [70]) that was origi-
nally isolated and characterized as a plasma membrane
protein with high affinity for the globular ‘heads’ of
the complement component C1q, but was actually just
one of its diverse binding partners. gC1qR is synthe-
sized with an N-terminal mitochondrial targeting
sequence that is cleaved after import into mitochon-
dria. The matrix location of this protein is firmly
established [71–73]; however, it is also found in other
cellular compartments [74]. In humans, it is encoded
by the C1qBP gene [75]. The function of this protein
in mitochondria is not known. gC1qR is an evolution-
arily conserved eukaryotic protein. Homologous genes
have been identified in a number of eukaryotic species,
ranging from fungi to mammals [74], compatible with
its potential role in PTP as the latter is also ubiquitous
among species. Its mitochondrial location and unique
structural features make gC1qR protein a highly plau-
sible candidate for the role of PTP channel, as dis-
cussed below.
Structural features of gC1qR as related
to PTP and Ca
2+
uniporter
There are striking structural features of this protein
that make it perfect for the roles of PTP channel and
calvectin-like ‘Ca
2+
uniporter’. The putative role of

gC1qR in PTP formation was suggested previously
[76], but this hypothesis did not attract much interest,
mostly due to then dominant view that the PTP is
formed by ANT, which has now been disproved [67].
gC1qR is a doughnut-shaped trimer with an outer
diameter of approximately 7.5 nm, a mean inner
diameter of approximately 2 nm, and a thickness of
approximately 3 nm. Each monomer consists of seven
consecutive b-strands forming a highly twisted antipar-
allel b-sheet. The channel wall is formed by the
b-sheets from all three subunits. This makes gC1qR a
potential target for cyclophilin D, a well-known modu-
lator of PTP sensitivity to Ca
2+
[55,58–60,63–66]. The
latter belongs to a class of enzymes called peptidyl-
prolyl-cis ⁄ trans-isomerases that act upon prolines in
b-sheets, resulting in a conformation change in the
target protein. The gC1qR is a very acidic protein with
a highly asymmetric negative charge distribution on
the protein surface. One side of the doughnut and the
inside portion of the channel possess a high number of
negatively charged residues; the opposite side is much
less negatively charged. These features permit the
gC1qR trimer to interact with other charged surfaces
such as phospholipid membranes or other proteins,
and the ability of gC1qR trimer to bind to the plasma
membrane surface is well documented [74]. Moreover,
these interactions are inherently sensitive to modula-
tion by divalent cations such as Mg

2+
and Ca
2+
,
which can bind to gC1qR and compensate its surface
charges. As gC1qR is acidic, its interactions with other
proteins may be weakened by increasing the acidity of
the medium. It is well known that PTP is inhibited at
low pH, probably due to release of cyclophilin D from
its putative binding site on the PTP [77]. Thus, it is
not unreasonable to suggest that gC1qR has a putative
binding site for cyclophilin D. Each monomer of
gC1qR has one cysteine at residue 186 (Cys186). This
residue does not form inter-chain disulfide bonds
between the monomers of a single gC1qR trimer [74].
However, under oxidative conditions, it forms a disulfide
bond between monomers of different gC1qR trimers,
thereby forming a hexameric structure consisting of two
Mitochondrial Ca
2+
sequestration system A. A. Starkov
3658 FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS
trimers. This complex has a much higher hydrody-
namic radius and altered ligand-binding properties
[78]. Lastly, a very important feature of the gC1qR tri-
mer is that its inner channel is very large (approxi-
mately 2 nm diameter in the compact trimeric crystal),
allowing easy passage of solutes with molecular mass
0.4–3 kDa [76], irrespective of their nature and electric
charge, which is compatible with the necessary PTP

channel properties. Moreover, the primary sequence of
gC1qR predicts three putative N-linked glycosylation
sites, and the protein was indeed found to be strongly
glycosylated [70]. Considering that gC1qR has to be
present at the cytosolic side of the inner mitochondrial
membrane (see below) for its many activities to be pos-
sible, the presence of glycosyl residues should render it
a target for ruthenium red binding, as it would be
expected form a putative Ca
2+
uniporter.
Interaction of gC1qR with pro-apototic
and other Ca
2+
-dependent cell signaling
cascades
Both the mitochondrial PTP and Ca
2+
uniporter are
implicated in so many pathological and physiological
scenarios that it would be virtually impossible for the
proteins involved in these systems to avoid multiple
interactions with other signaling and regulatory pro-
teins. It has been shown that gC1qR is a partner of
the pro-apoptotic protein Hrk [79], a mammalian
BH3-only protein. Multiple lines of experimental evi-
dence verified a specific interaction and co-localization
of Hrk and gC1qR, both of which depended on the
presence of the highly conserved C-terminal region of
gC1qR. Hrk-induced apoptosis was suppressed by

expression of gC1qR mutants lacking the N-terminal
mitochondrial signal sequence (gC1qR
74–282
) or the
conserved C-terminal region (gC1qR
1–221
), which inhi-
bit competitive binding of Hrk to the gC1qR protein
and disrupt the channel function of gC1qR, respec-
tively [79]. Another recently discovered pro-apototic
protein, smARF, also binds to mitochondrial gC1qR.
This protein is known to induce autophagic cell death.
gC1qR physically interacts with both human and mur-
ine smARF, and co-localizes with them to the mito-
chondria. Remarkably, knock-down of gC1qR levels
significantly reduced the steady-state levels of smARF
by increasing its turnover. As a consequence, the abil-
ity of ectopically expressed smARF to induce auto-
phagy was significantly reduced. gC1qR stabilizes the
smARF [80]. Mitochondrial gC1qR has also been
shown to be a substrate for ERK and an integral part
of the MAP kinase cascade [81]. It is also a general
protein kinase C (PKC)-binding protein [70]; it binds
to and regulates the activity of PKC isoforms PKC-a,
PKC-f, PKC-d, and PKC-l (the latter being constitu-
tively associated with gC1qR at mitochondrial
membranes) without being their substrate [82]. Several
lines of evidence suggest that mitochondrial PKC may
directly regulate PTP status, at least in heart [83], and
the involvement of PTP in cell apoptosis is suggested

in so many publications that it is difficult to provide a
key reference. The most recent data strongly linking
gC1qR to Ca
2+
-related mitochondrial dysfunction and
apoptosis was obtained by Chowdhury et al. [84], who
demonstrated that constitutively expressing gC1qR in
a normal murine fibroblast cell line induced growth
perturbation, swelling and derangements of cristae in
cell mitochondria, release of cytochrome c and forma-
tion of apoptosome complexes. They also showed that
mitochondrial dysfunction was due to a gradual
increase in ROS generation in cells over-expressing
gC1qR. Together with ROS generation, they found an
increased Ca
2+
influx in mitochondria, resulting in a
decreased membrane potential and severe inhibition of
the respiratory chain complex I [84].
Fig. 2. Proposed model of CA
2+
uniporter and PTP assembly. The
‘protomers’ (flat disks) of a putative protein forming the ‘Ca
2+
uni-
porter’ (‘U’) and PTP (e.g. gC1qR as discussed in the text) are pres-
ent in the intermembrane space, the inner membrane and the
matrix of mitochondria, but the ‘Ca
2+
uniporter’ consisting of

IM- and matrix-located protomers, is not assembled to its fully
active form. When a threshold amount of Ca
2+
is reached, a few
protomers migrate from the intermembrane space to the IM and
bind to the protomer located in the IM. This creates a fully func-
tional ‘Ca
2+
uniporter’. Such binding does not occur in the presence
of ruthenium red, which blocks it by interacting with the glycosyl
residues of IM-embedded protomers. Accumulated Ca
2+
and phos-
phate bind to an unidentified ‘nucleation factor’ (‘n.f.’) that prevents
the formation of crystalline Ca
2+
-phosphate precipitates. Upon
prolonged accumulation of Ca
2+
, the storage capacity of this ‘nucle-
ation factor’ is exceeded, and the Ca
2+
concentration in the matrix
and the intermembrane space rises above the threshold for PTP
assembly, thereby triggering formation of a larger multi-component
PTP channel (see text for further details).
A. A. Starkov Mitochondrial Ca
2+
sequestration system
FEBS Journal 277 (2010) 3652–3663 ª 2010 The Author Journal compilation ª 2010 FEBS 3659

How could gC1qR participate in both
the PTP channel formation and in Ca
2+
uniport?
On the basis of the structural features of gC1qR, a
novel mechanism of PTP formation and Ca
2+
trans-
port can be proposed that involves the same protein in
both systems (Fig. 2). According to this model, gC1qR
‘protomers’ are present in the intermembrane space,
the inner membrane and the matrix of mitochondria,
but the ‘Ca
2+
uniporter’, consisting in this case of IM-
and perhaps matrix-located gC1qR protomers, is not
assembled to its fully active form. When a threshold of
Ca
2+
is reached, a few protomers of gC1qR migrate
from the intermembrane space to the IM and bind to
the protomer located in the IM. This creates a fully
functional Ca
2+
uniporter. The binding does not occur
in the presence of ruthenium red, which blocks it by
interacting with the glycosyl residues of IM-embedded
protomers. Prolonged accumulation of Ca
2+
results in

its concentration in the matrix and the intermembrane
space exceeding another Ca
2+
threshold, triggering the
formation of a larger, multi-component PTP channel
(Fig. 2). Both the Ca
2+
transport system and the PTP
have to be pre-assembled for their full activity, and
our hypothesis is that they are two stages of the same
process of Ca
2+
-dependent assembly of gC1qR pro-
tomers, perhaps in co-operation with some other regu-
latory proteins. The PTP channel in the IM may be
formed by a gC1qR multimer comprising several (e.g.
three in Fig. 2) identical gC1qR trimers stacked onto
each other. Formation of the multimer means that the
structure acquires sufficient hydrophobicity to move
into the IM and form a transmembrane channel. For-
mation of this structure is caused by Ca
2+
accumula-
tion in the matrix, but not necessarily an increase in
free Ca
2+
concentration. For example, gC1qR trimers,
which are inherently capable of binding Me
2+
ions,

may initially be sequestered by another Me
2+
-binding
protein in the matrix as a gC1qR*Mg
2+
complex, and
the Me
2
+
-binding protein may also be capable of serv-
ing as a nucleation factor catalyzing the formation of
Ca
2+
-phosphate precipitates. When the latter accumu-
late, they displace gC1qR from the complex, thereby
priming it to PTP. The next step may be a change in
gC1qR conformation that would increase the probabil-
ity of its interaction with another gC1qR trimer to
form a hexamer. This conformational change may be
facilitated by binding to cyclophilin D. The last step is
a further Ca
2+
-dependent change in conformation of
this newly formed hexameric gC1qR–cyclophilin D
complex to allow it to translocate into the IM and
form the PTP. In this model, chelating free Ca
2+
by
EGTA would force the structure to leave the IM, sup-
pressing channel formation, but will not reverse the

entire process because it could not quickly remove the
Ca
2+
-phosphate precipitates that have already formed.
Therefore, the pore-forming complex will remain
primed and ready for repeated cycles of PTP opening
and closing. On the other hand, Cys186 mediated for-
mation of disulfide bridges between the two gC1qR tri-
mers would render the channel structure permanent
and insensitive to modulation by Ca
2+
or cyclophi-
lin D, thereby producing an ‘unregulated’ pore. This
PTP model can easily accommodate most if not all
known data on PTP activation and regulation, includ-
ing its sensitivity to a wide variety of chemically dis-
similar compounds and even to the changes in the
conformation of major IM proteins that are capable of
modifying the surface charge of the IM, such as ANT.
Concluding remarks
Obviously, this model is highly speculative as there are
no data that directly support its key features. How-
ever, there are three predictions about this mechanism
that can be verified experimentally. First, gC1qR has
to physically move into the IM to form a PTP, i.e.,
upon accumulation of Ca
2+
and phosphate, the distri-
bution of free gC1qR between the mitochondrial com-
partments should change dramatically towards the IM,

preceding opening of the PTP. Second, knocking out
the gC1qR protein should prevent Ca
2+
-induced PTP
formation or at least severely increase its Ca
2+
thresh-
old. Third, antibodies against gC1qR should inhibit
Ca
2+
uptake in mitoplasts. We are currently trying to
verify these predictions experimentally.
Acknowledgement
This work was supported by National Institutes of
Health grant number NS065396 to A.S.
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